Detection of Ki-ras mutations in tissue and plasma samples of patients with pancreatic cancer using PNA-mediated PCR clamping and hybridisation probes

Däbritz, Jan; Hänfler, Joachim; Preston, Roman; Stieler, Jens; Oettle, Helmut

doi:10.1038/sj.bjc.6602319

Cited by 77 publications

(51 citation statements)

References 39 publications

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“…PNA -PCR clamp method can rapidly (within 2 h) detect K-Ras mutations using a low quantity of DNA. By contrast to other PNA studies, we used wild-type fluorescent-labelled hybridisation probes allowing rapid and high-sensitive detection of all the K-Ras mutations (Sun et al, 2002;Chen et al, 2004;Taback et al, 2004;Däbritz et al, 2005;Luo et al, 2006;Miyake et al, 2007). Only one pair of primers and one pair of probes are required to detect all possible mutations in codons 12 and 13 of the K-Ras gene.…”

Section: Discussionmentioning

confidence: 99%

“…Using this method, c-Kit point mutations have been detected in skin biopsy samples from patients with urticaria pigmentosa (Sotlar et al, 2003). Such a technique has been applied to search for K-Ras mutations in various tumour samples (Sun et al, 2002;Chen et al, 2004;Taback et al, 2004;Däbritz et al, 2005;Luo et al, 2006;Miyake et al, 2007). This method was also used to detect EGFR mutations in NSCLC (Nagai et al, 2005;Soh et al, 2006;Sutani et al, 2006;Tanaka et al, 2007;Miyanaga et al, 2008).…”

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confidence: 99%

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Detection of K-Ras mutations in tumour samples of patients with non-small cell lung cancer using PNA-mediated PCR clamping

et al. 2009

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Non-small cell lung cancers (NSCLC), in particular adenocarcinoma, are often mixed with normal cells. Therefore, low sensitivity of direct sequencing used for K-Ras mutation analysis could be inadequate in some cases. Our study focused on the possibility to increase the detection of K-Ras mutations in cases of low tumour cellularity. Besides direct sequencing, we used wild-type hybridisation probes and peptide-nucleic-acid (PNA)-mediated PCR clamping to detect mutations at codons 12 and 13, in 114 routine consecutive NSCLC frozen surgical tumours untreated by targeted drugs. The sensitivity of the analysis without or with PNA was 10 and 1% of tumour DNA, respectively. Direct sequencing revealed K-Ras mutations in 11 out of 114 tumours (10%). Using PNA-mediated PCR clamping, 10 additional cases of K-Ras mutations were detected (21 out of 114, 18%, P <0.005), among which five in samples with low tumour cellularity. In adenocarcinoma, K-Ras mutation frequency increased from 7 out of 55 (13%) by direct sequencing to 15 out of 55 (27%) by clamped-PCR ( P <0.005). K-Ras mutations detected by these sensitive techniques lost its prognostic value. In conclusion, a rapid and sensitive PCR-clamping test avoiding macro or micro dissection could be proposed in routine analysis especially for NSCLC samples with low percentage of tumour cells such as bronchial biopsies or after neoadjuvant chemotherapy.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Detection of K-Ras mutations in tumour samples of patients with non-small cell lung cancer using PNA-mediated PCR clamping

et al. 2009

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“…14 This approach significantly increases the sensitivity of mutation detection. PCR products were re-amplified with nested primers and sequenced for confirmation.…”

Section: Molecular Studiesmentioning

confidence: 99%

Development of monocytosis in patients with primary myelofibrosis indicates an accelerated phase of the disease

et al. 2013

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Primary myelofibrosis is a type of chronic myeloproliferative neoplasm characterized by progressive bone marrow failure with worsening cytopenia and in a subset of patients, progression to acute leukemia. Published data in patients with myelodysplastic syndromes have shown that the development of monocytosis in the course of myelodysplastic syndromes is associated with a poor prognosis. A similar occurrence has been only sporadically reported in patients with primary myelofibrosis. Over a period of four years we identified 10 out of 237 cases of primary myelofibrosis who developed persistent absolute monocytosis (41 Â 10 9 /l) during the course of disease (5 men and 5 women; median age/range: 68 years/52-82). Monocytosis developed at a median interval of 42 months from diagnosis (range: 1-180) and persisted for a median period of 23 months (range: 2-57). Five patients died after developing monocytosis (range: 20-188 months) and two experienced worsening disease and became transfusion dependent. Monocytosis was associated with increased white blood cells, decreased hemoglobin, decreased platelet count, and the presence of circulating blasts. In three cases, bone marrow biopsies after the onset of monocytosis showed marked myelomonocytic proliferation with morphological shifting from a typical primary myelofibrosis marrow appearance to aspects compatible with an overt 'secondary' chronic myelomonocytic leukemia. Before the development of monocytosis, 5 of 10 patients carried the JAK2V617F mutation; five patients showed karyotypic alterations. No change in JAK2 mutational status or cytogenetic evolution were associated with the development of monocytosis. Four of nine patients analyzed showed KRAS mutation in codon 12 or 13 with low allele burden. This is the first study correlating monocytosis developing in primary myelofibrosis patients with bone marrow morphology, laboratory data, molecular analysis and clinical follow-up. Development of monocytosis in patients with established primary myelofibrosis is associated with rapid disease progression and these patients should be considered as a high-risk group associated with short survival.

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“…The sequence of the PCR primers, probes, and PNA of the real-time PCR assay adapted from Dä britz et al 17 were as described, except for the acceptor probe, which had the wild-type sequence 5Ј-LC Red640-TTGC-CTACGCCACCAGCTCCAA-3Ј. The PNA sequence was 5Ј-CCTACGCCACCAGCTCC-3Ј.…”

Section: Primers Probes and Peptide Nucleic Acidmentioning

confidence: 99%

High-Fidelity DNA Polymerase Enhances the Sensitivity of a Peptide Nucleic Acid Clamp PCR Assay for K-ras Mutations

Gilje

Heikkilä

Oltedal

et al. 2008

The Journal of Molecular Diagnostics

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Sensitive detection of tumor-specific point mutations is of interest in both the early detection of cancer and the monitoring of treatment at a molecular level. Recently , peptide nucleic acid (PNA) clamp real-time PCR has provided a time-sparing and sensitive method for the detection of mutations in the presence of a large excess of wild-type DNA. We present the first report that the sensitivity of PNA clamp PCR is limited by the low fidelity of Taq DNA polymerase. Replication errors introduced by Taq polymerase in the PNA-binding site were amplified during PCR due to the resulting mismatches between PNA and DNA. To reduce the frequency of polymerase-induced errors , we developed a PNA clamp PCR assay for the detection of mutations in codons 12 and 13 of the K-ras gene based on a high-fidelity DNA polymerase. The sensitivity of our assay increased approximately 10-fold , significantly detecting mutant DNA diluted 20 ,000-fold in wild-type DNA (P ‫؍‬ 0.025) , compared with its detection at 2000-fold dilution (P ‫؍‬ 0.039) when Taq polymerase was used. Our data suggest that the replication errors caused by Taq polymerase must be taken into consideration for PNA clamp PCR and for other methods based on selective PCR amplification, and that these assays can be enhanced by highfidelity DNA polymerases.

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Detection of Ki-ras mutations in tissue and plasma samples of patients with pancreatic cancer using PNA-mediated PCR clamping and hybridisation probes

Cited by 77 publications

References 39 publications

Detection of K-Ras mutations in tumour samples of patients with non-small cell lung cancer using PNA-mediated PCR clamping

Detection of K-Ras mutations in tumour samples of patients with non-small cell lung cancer using PNA-mediated PCR clamping

Development of monocytosis in patients with primary myelofibrosis indicates an accelerated phase of the disease

High-Fidelity DNA Polymerase Enhances the Sensitivity of a Peptide Nucleic Acid Clamp PCR Assay for K-ras Mutations

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